The present invention relates to a kit and to an immunometric assay method using this kit for the determination of surface antigens characteristic of populations or subpopulations of cells. The immunometric assay method and the corresponding kit are also intended for the determination of the cells themselves via the determination of their surface antigens. These determinations are applicable to diagnosis.
Knowledge of the antigens or markers on the cell surface has made enormous advances with the development of lymphocyte hybridization and the discovery of monoclonal antibodies by KOEHLER and MILSTEIN (Nature, 1975, 256, 495-497). In particular, monoclonal antibodies have made it possible to reveal and analyze membrane antigens or surface markers of cells of the widest possible variety of origins. These markers (or antigens) can be of different kinds: proteins, glycoproteins or glycolipids. The characterizations sought therefore apply mainly to tissue or organ markers, to markers of states of differentiation or activation of normal cells and to the identification or typing of normal or cancerous cells. A particularly important field of application is the study of the cell lines of he mopoiesis (erythrocyte, megakaryocyte, granulocyte, monocyte, lymphocyte).
Thus, for example, monoclonal antibodies have made it possible to specify the respective surface characteristics of T and B lymphocytes. The corresponding markers, by themselves or in combination, identify stages of differentiation and functional specialization of the lymphocytes. By international convention, the surface markers of human leukocytes have been classified in differentiation groups or differentiation classes (CD) defined by the IUIS-WHO subcommittee, 1984, and described in Bulletin of the World Health Organization, 1984, 62(5), 813-815.
The identification of these markers which has been made possible by monoclonal antibodies has provided access to their structure and their biological functions. For example, the molecules of the CD4 and CD8 markers participate in leukocyte adhesion functions and are present on the surface of T lymphocytes with an auxiliary and inductive function (CD4 marker) or, respectively, with a cytotoxic and suppressive function (CD8 marker).
With this knowledge established, it has been possible to use these markers, by virtue of the antibodies which recognize them, for diagnosing and following up a variety of pathologies including, in particular, malignant hemopathies (leukemias, lymphomas, etc.) and states of dysfunction of the immune system (autoimmune diseases, congenital or acquired immune deficiencies such as AIDS, etc.) (BRETON-GORIUS and VAINCHENKER, Le Biologiste, 1987, XXI, no. 167, 63-70; SHAW, Immunology Today, 1987, 8(1), 1-3).
Monoclonal antibodies are now irreplaceable tools of clinical biology applied to cell analyses.
Cell counting methods exist which use the marking of their surface antigens, but these methods are often lengthy, laborious and difficult to carry out and their results are sometimes random.
The known methods used for measuring the normal or modified expression of antigens on the surface of the cell can be separated into two groups. In the first group, the antigens are measured with the aid of complex and specialized laboratory equipment based on flow cytometry (see, in particular, PONCELET et al., J. Immunol. Methods, 1985, 85, 65-74) or quantitative microscopy techniques (POULTER et al., J. Immunol. Methods, 1987, 98, 227-234). These methods for the evaluation of cell antigens are based on the measurement of signals provided by anticell antibodies coupled directly or indirectly to a reagent labeled with fluorescent substances (or fluorochromes) such as fluorescein isothiocyanate or rhodamine isothiocyanate, or with enzymes such as peroxidase or alkaline phosphatase. The use of these fluorescent or enzyme reagents in association with appropriate washing steps then leads to the appearance of fluorescences or colorations which are strictly limited to the cell membranes and do not diffuse into the surrounding medium. Common use of these methods in the laboratory is still restricted by the need for specialized and expensive equipment (a fluorescence microscope which may or may not be associated with an image analyzer, a cryostat and a flow cytometer). Moreover, the analysis and interpretation of th immunolabeling of cells by these processes demand the competence of a specialist in cytology.
A second group of methods for the measurement of antigens is based on the quantitative evaluation of the markers of the overall cell population. These methods make it possible to measure antigens either by direct labeling or by indirect labeling, the latter most frequently being carried out in two, three or four steps. In all cases, the reagent employed in the last labeling step carries a probe which is either of isotopic character, for example iodine 125, for a determination of the radioimmunometric type (BROW et al.,J. Immunol. Methods, 1979, 31, 201; STOCKER and HEUSSER, J. Immunol. Methods, 1979, 26, 87-95), or an enzyme for a determination of the enzyme immunometric type, most frequently peroxidase, alkaline phosphatase or beta-galactosidase (VAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 109-116; MORRIS, Transplantation, 1983, 36(6), 719; BAUMGARTEN, J. Immunol. Methods, 1986, 94, 91-98).
The methods in this last group are rather inconvenient, laborious and risky to apply because of the need to ash and centrifuge the cell material many times; it is sometimes necessary to take a sample of the colored medium resulting from the enzyme reaction in order to carry out the final spectrophoto-metric measurement; finally, chemical fixation of the cells, which is used in most cases, causes the irreversible destruction of certain antigens which are particularly sensitive to the customary chemical binding agents such as glutaraldehyde or methanol (DROVER et al., J. Immunol. Methods, 1986, 90, 275-281).
It is known in the literature that an antigen carrying several antigenic determinants, i.e. several epitopes, can be determined by fixing this antigen via one of its epitopes using an antibody immobilized on a solid support, and by binding, to another epitope of the antigen, another antibody carrying an enzyme or radioisotopic marker enabling the determination to be carried out.
This kind of technique, which is often referred to as the sandwich technique, is described especially in French Patent 2 487 983, French Patent 2 500 166 and European Patent Application 119 736. None of these documents describes the application of this technique to whole cells, even though the word xe2x80x9ccellxe2x80x9d is sometimes included in the list of antigens to which the process applies.
In the above patents, the various antigens forming the subjects of the Examples described are in all cases solely protein molecules soluble in water and physiological liquids, such as tumoral markers, enzymes or hormones in the bloodstream. On the other hand, it is clear that a cell is not a molecule and differs therefrom at least by being considerably larger and by the fact that it is not soluble in physiological media. Thus,the sandwich technique has so far never been applied to whole cells.
Furthermore, the immunocapture of cells on a solid support is described in International Patent Application 86/02091, in which the object is to remove undesirable cells from samples of bone marrow intended for transplantations. In the said patent application, capture of the cells is effected on floating microbeads and requires that the antibody used be bound to the solid support by a complex macromolecular structure, called a network-relay, which is capable of ensuring a preferential orientation of the antibody relative to that of the corresponding cell antigen. The said patent application gives no indication of an application of the technique to the quantitative determination of an antigen.
The immunocapture of cells is also described in International Patent Application 84/03151 for an analytical application. In the said patent application, the object is to identify the tissue groups to which the examined cells belong (this operation generally being called HLA typing). The cells are captured by means of antibodies arranged according to a particular geometry on very specialized supports (microscope cover glasses). The results are obtained simply by visual observation of the support and produce xe2x80x9call or nothingxe2x80x9d responses. Thus the cell immunocapture systems described hitherto do not lead to analytical applications permitting the quantitative determination of an antigen expressed at the membrane of certain cells. Furthermore, all these systems may lack specificity because they are based on recognition of the cells by a single antibody.
The method of determination forming the subject of the present invention has considerable advantages compared with all the techniques known and used in the prior art, since it permits the quantitative measurement of any antigen of a cell population in a single analysis step. This determination is carried out on cells which have not undergone any chemical or physical intervention and which are in their state of physiological integrity. Furthermore, the method of determination according to the invention has the characteristics of very high specificity which are inherent in the double immunological recognition systems involving 2 different antibodies specific for 2 different antigens carried by the same cell. This method is simple, rapid and reproducible. It is totally suitable for the analysis of a large number of samples, which enables it to be used for diagnostic purposes in clinical biology laboratories handling these large numbers.
The present invention thus relates to a kit for the determination of at least one surface antigen characteristic of a cell population or subpopulation, which comprises the following components:
a) a solid support to which one or more monoclonal antibodies are fixed by covalent bonding or physical adsorption, the said monoclonal antibodies being directed against surface antigens of the cell population examined, other than the said characteristic antigen, and being intended to immobilize, on the support, the cells which include those of the subpopulation carrying the antigen to be determined;
b) at least one solution comprising a monoclonal antibody specific for the said antigen characteristic of the cell population or subpopulation carrying the antigen to be determined, which is labeled with a radioisotopic probe or an enzyme probe; and
c) in the case of monoclonal antibodies labeled with an enzyme probe, a developer for the enzyme, namely one or more solutions containing the substrate for the enzyme and, if necessary, one or more reagents necessary for developing the activity of the enzyme.
The term xe2x80x9ccellxe2x80x9d as used in the present specification and in the claims which follow includes human cells, animal cells, the cells of protozoans and the cells of microorganisms (bacteria or fungi). As regards blood cells, the present invention includes the nucleate formed elements, such as the leukocytes, and the a nucleate formed elements, such as the erythrocytes or platelets.
The method of determination according to the invention applies to whole cells, i.e. non-lyzed cells.
These cells have not undergone any physical or chemical intervention and they are used in a state of complete physiological integrity. This situation constitutes the best guarantee of integrity of the membrane markers chosen as targets for determination.
As the solid support it is possible to use any device suitable for the handling of cell suspensions, and preferably tubes, particulate magnetic supports or rigid or flexible microtiter plates made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose, which contain microwells. The monoclonal antibodies intended for immobilization of the cells can be fixed to the solid supports either by covalent chemical bonding or by physical adsorption according to conventional methods such as those disclosed by STOCKER and HEUSSER J. Immunol. methods, 1979, vol. 26, p. 87-95. Advantageously, the support is saturated with a protein beforehand.
According to the invention, the monoclonal antibody or antibodies fixed to the solid support must permit the immunocapture of the cell population which includes the cell population or populations carrying the antigens to be determined. When this population consists of human cells, the preferred monoclonal antibodies for immunocapture are the anti-class I HLA antibodies which are specific for the common part of the HLA-A, -B and -C antigens present on the leukocytes and numerous other cell lines of the organism. Of these antibodies, the one called S-class I, marketed by BIOSYS, is particularly preferred.
In other cases where the cells examined are human cells and in all cases where these cells are not human cells, monoclonal antibodies appropriate to the type of cells examined can also be used for the immunocapture according to the invention.
The expression xe2x80x9ca monoclonal antibody labeled with a radioisotopic probexe2x80x9d means that the monoclonal antibody carries, either on a component inherent in its structure, for example the constituent tyrosine residues, or on an appropriate radical which has been attached thereto, a radioactive isotope which enables it to be determined by counting the radioactivity associated therewith.
The expression xe2x80x9ca monoclonal antibody labeled with an enzyme probexe2x80x9d means that the monoclonal antibody is coupled to an enzyme which, when associated with the use of appropriate reagents, permits quantitative measurement of this monoclonal antibody.
The substrate and the reagents are chosen so that the final product of the reaction or reaction sequence caused by the enzyme, involving these substances, is:
either a colored or fluorescent substance which diffuses into the liquid medium surrounding the cells and which is the object of the final spectrophoto-metric or, respectively, fluorimetric measurement,
or an insoluble colored substance which deposits on the cells and the walls to which they are fixed, and which can be the object either of photometric measurement by reflection or of visual evaluation, if appropriate against a range of standard shades.
As an additional component, the assay kit can contain a buffer solution intended for washing the solid support after immobilization and labeling of the cells with the antibody or antibodies carrying the chosen probe.
As additional components, the assay kit can also contain the samples necessary for standardization and quality control of the determination.
The present invention further relates to a process for the immunometric assay of the surface antigens of a cell population or subpopulation, the said process comprising:
a single step for the specific immobilization or immunocapture of a cell population on the solid support using one or more monoclonal antibodies fixed to the said support beforehand by covalent bonding or by physical adsorption and capable of recognizing an antigen present on the surface of the cells, other than the antigen to be determined, and, simultaneously, the direct labeling of the surface antigen to be determined, belonging to the immobilized cell population or to one of its subpopulations, with a monoclonal antibody specific for this antigen to be determined, the said monoclonal antibody carrying a radioisotopic or, alternately, enzyme probe;
an incubation period to allow the simultaneous immuno-capture and labeling to take place;
the washing of the solid support to remove the nonimmobilized undesirable cells and the excess of monoclonal antibody carrying the radioisotopic or enzyme probe; and
the actual determination of the antigen to be determined in the labeled cell population or subpopulation by counting the fixed radioactivity or, alternately, after treatment of the medium with the substrate for the enzyme and, if necessary, one or more appropriate auxiliary reagents, by photometric measurement by transmission or reflection, or measurement of the fluorescence emission.
The assay kit and the immunometric assay process according to the invention are preferably applied to the determination of the surface antigens of the formed elements of human blood, especially the leukocytes and more particularly the lymphocytes, the T lymphocytes, the T4 lymphocytes, the T8 lymphocytes and the B lymphocytes, as well as the granulocytes, the monocytes and the blood platelets.
Another preferred application is the determination of the surface antigens of pathogenic micro-organisms, for example Candida albicans. 
The assay kit and the immunometric assay process according to the invention are also particularly useful for the determination of the surface antigens of tumoral cells, especially those of cancers of the urinary system and those of malignant hemopathies.
The assay kit and the immunometric assay process according to the invention make it possible to measure signals (absorbed or emitted light or radioactivity) which depend both on the number of cells present in the cell population examined and on the density of the antigen measured on the surface of these cells. Measurement of these signals permits quantitative evaluation of the total number of molecules of this antigen which are carried by the cell population or subpopulation examined, whether this antigen has a structural or functional role.
For example, in the case of the leukocyte markers of particular importance in hematology, it is known that, in the majority of situations in healthy subjects, the mean value of the antigen density does not vary substantially between samples for one and the same cell population, so there is a good correlation between the cytological count of the cells carrying the antigen in question and the signal measured according to the invention, which is proportional to the total number of antigen molecules present in the sample examined. Conversely, in some pathological states, the density of the surface antigens may vary for one and the same cell population without a notable variation in the number or proportion of the positive cells. Such pathological immunometric assay process according to the invention or by using the kit of the present invention than they would be if a kit of the present invention than they would be if a conventional cytological counting procedure were used.
Another application of the invention becomes apparent if a microtiter plate is chosen as the solid support. The assay kit and the immunometric assay process according to the invention can then advantageously be used for the determination, on a single plate, of a series of surface antigens characteristic of various subpopulations making up the cell population examined.
For this application, it is possible on the one hand to take ready-to-use microtiter plates to which one or more monoclonal antibodies capable of retaining all the cells of the population examined have been fixed beforehand, and on the other hand to have a series of monoclonal antibodies coupled to an appropriate probe and each specific for an antigen characteristic of one of the subpopulations to be evaluated. Thus, in a single manipulation and on one and the same support, it is possible to carry out the quantitative determination of all the antigens necessary for characterization of the chosen subpopulations.
This application of the present invention is illustrated by the characterization of the antigenic equipment of cells of interest in clinical biology. A first case is represented by the determination of the tissue groups characterizing a given individual, which is conventionally known as HLA typing.
A second case is represented by the typing of tumoral cells, in particular for patients afflicted by malignant hemopathies such as leukemias or lymphomas. This diagnostic examination, which is practiced systematically, consists in characterizing the type and origin of the patient""s tumoral cells by the presence or absence on these cells of a series of suitably chosen surface antigens.
Use of the kit according to the invention, which contains a microtiter plate on which one or more monoclonal antibodies capable of fixing all the cells of the population examined have been fixed beforehand, and solutions of different monoclonal antibodies labeled with an enzyme or radioisotopic probe and each specific for an antigen present on the tumoral cells, permits identification and quantitative evaluation of the antigens characteristic of the patient""s tumoral cell population and thus makes it possible to associate them with one of the major groups of clinically characterized cancers, especially malignant hemopathies. Application of the process according to the invention thus makes it possible to carry out the qualitative and quantitative examination of the antigenic phenotyope of tumoral cells rapidly and on a single support.
A case which may be mentioned as an illustration of another application of the invention is that of the human T lymphocytes, for which there are in particular subpopulations of cells: the lymphocytes characterized by the presence of the CD4 marker, which will be called positive T4 lymphocytes or, more simply, T4 lymphocytes, and the lymphocytes characterized by the presence of the CD8 marker, which will be called positive T8 lymphocytes (or T8 lymphocytes).
Measurement of the numerical ratio T4/T8 is of great diagnostic interest in clinical biology. In fact, it is known that modifications of the T4/T8 ratio appear in various complaints of the immune system, such as dysimmune diseases, chronic infectious diseases, viral infections and, in particular, HIV complaints (AIDS virus).
The assay kit and the immunometric assay process according to the invention can be used for the determination of antigens universally characteristic of the T lymphocyte population and/or antigens characteristic of the T4 and T8 lymphocyte subpopulations. In this case, the T lymphocytes of the sample examined are specifically immobilized on a solid support and, simultaneously, the surface antigens of the T4 lymphocytes are labeled directly with an anti-CD4 monoclonal antibody carrying a radioisotopic or enzyme probe; in the same way, the surface antigens of the T8 lymphocytes are labeled directly with an anti-CD8 monoclonal antibody carrying an appropriate probe.
The total T lymphocytes are preferably determined using an anti-CD7 monoclonal antibody (also called anti-T2 monoclonal antibody) carrying an appropriate probe.
Specific immobilization of the T lymphocytes of the sample is preferably carried out using one or more monoclonal antibodies which are capable, by themselves or in combination, of recognizing all the T cells of the sample, this being the case of the anti-common leukocyte (or anti-CD45) antibodies or antibodies which recognize the whole of the T population (called xe2x80x9cpan-Txe2x80x9d antibodies), such as the anti-CD2 (or anti-T11), anti-CD5 (or anti-T1) or anti-CD7 (or anti-T2) antibodies or other pan-T anti-bodies which have not yet been assigned to a differentiation class according to the WHO criteria.
The immunometric assay method of the invention can advantageously be used for the determination of antigens characteristic of the population of T lymphocytes and T4 and T8 lymphocytes on several parts of the same solid support. Measurement of the signals by radioactivity counting, photometric measurement by transmission or reflection or fluorescence measurement enables the numerical ratio CD4/CD8 to be calculated easily and directly.
In the same way, it is possible to determine, on one and the same solid support, the subpopulations called T lymphocytes and B lymphocytes which make up the whole of the lymphocyte line.
For example, a monoclonal antibody or a mixture of monoclonal antibodies specific for all the surface antigens of the T cells can be fixed by adsorption to the walls of the microwells. These monoclonal anti-bodies will enable the whole population of T cells of the sample examined to be immobilized in the microwells at a later stage. The plates prepared in this way can be lyophilized and stored, preferably at 4xc2x0 C. This step can be carried out on the industrial scale and it will thus be possible to have ready-to-use plates for the assay kits which can be applied either to the total T lymphocytes or to any subpopulation of T lymphocytes.
The samples containing the cells to be determined, which originate from the blood or from any appropriate biological liquidxe2x80x94normal or pathologicalxe2x80x94can be used as such or after preparation, especially by density gradient centrifugation according to the methods already known, and in particular in a high-density medium such as, for example, FICOLL-PAQUE marketed by Pharmacia. To determine the blood lymphocytes, the blood sample to be determined can also be treated with a so-called lysis buffer solution, which lyzes the erythrocytes.
It will be noted that particulate magnetic supports, notably magnetic beads are particularly appropriate to carry out the assay of a cell population or subpopulation contained in a biological liquid, without a previous preparation of this cell population or subpopulation from said biological liquid. For example, by using particulate magnetic supports, it is possible, according to the process of the invention to directly assay the formed elements of human blood from the complete blood or the urines.
Aliquots of the appropriate cell suspension are brought into contact with the solid support, for example in the microwells of a microtiter plate prepared beforehand, at the same time as the solution forming part of the assay kit, which contains the monoclonal antibody specific for the target cell population and carrying an appropriate probe, i.e. a radioisotopic or enzyme tracer. Thus a radioisotopic probe can be prepared for example by labeling the monoclonal antibody with iodine 125 or iodine 131, for example in the presence of chloramine T, by a known process (F. C. GREENWOOD, W. M. HUNTFR et al., Biochem. J., 1963, 89, 114); alternately, an enzyme probe can be prepared by conjugating the monoclonal antibody with an enzyme such as alkaline phosphatase, peroxidase, beta-galac-tosidase or acetylcholinesterase, by a described method (see, for example, M. O""SULLIVAN, Methods in Enzymology, 1981, 73, 147) or by a method based thereon. In some cases, in order to avoid certain disadvantages associated with the handling of radioactive substances and the limited shelf life of the reagents, enzymes will be used in preference to radioisotopic probes.
The incubation period, i.e. the time required for immobilization and simultaneous labeling of the cells, is preferably less than or equal to 1 hour. During this time, the solid support can be centrifuged, if necessary, in order to improve the immobilization of the cells. The solid support, for example the microtiter plate, is then washed to remove the non-fixed cells and at the same time the excess of monoclonal antibody carrying an enzyme or radioisotopic probe.
When a radioisotopic probe is used, for example iodine 125, the radioactivity associated with the cells is counted in a gamma counter according to any appropriate procedure and, for example, after solubilization of the cells with an alkaline solution (for example a sodium hydroxide solution) and recovery of the solution containing the radioactivity by means of an absorbent buffer.
When an enzyme probe is used on the monoclonal antibody, the appearance of a colored or fluorescent product is brought about by adding, to the solid support to which the cell population carrying the antigen to be determined has been fixed, a solution containing the substrate for the enzyme and one or more auxiliary reagents such that the reaction product which is finally obtained is either a colored product soluble in the medium, or an insoluble colored product, or a soluble fluorescent product, as explained earlier. The light signal coming from the samples treated in this way is then measured with the equipment appropriate to each case, i.e. a transmission or reflection photometer or, respectively, a fluorimeter. When the solid support is a microtiter plate, the light signal can be read sequentially in all the wells of one and the same plate by means of automated readers commonly used in biology laboratories, such as the Titertek plate reader or the Fluoroscan plate reader for the spectrophotometric or, respectively, fluorometric readings.
When alkaline phosphatase is used as the enzyme probe, this enzyme is coupled to the monoclonal antibody according to the method proposed by Boehringer Mannheimxe2x80x94Biochemica. The preferred substrates for this enzyme are paranitrophenyl phosphate for a spectrophotometric final reading, 4-methylumbelliferyl phosphate for a fluotimetric reading or 5-bromo-4-chloroindol-3-yl phosphate for obtaining an insoluble colored reaction product. Likewise, beta-galactosidase can be used as the enzyme probe, in which case the preferred substrates will be orthonitrophenyl beta-D-galactopyranoside or 4-methylumbelliferyl beta-D-galactopyranoside.
The monoclonal antibodies can preferably be coupled to peroxidase. In this case, the coupling process is derived from that described by M. B. WILSON and P. K. NAKANE in Immunofluorescence and Related Staining Techniques, edited by W. Knapp, K. Kolubar and G. Wicks, Elsevier/North Holland, Amsterdam, 1978, p. 215-224. The modifications which have been introduced by comparison with the initial protocol for preparation of the enzyme conjugate concern the following points:
the molar ratio peroxidase/antibody is equal to 3 as opposed to 2 in the protocol, and
oxidation of the carbohydrate units of the peroxidase is less harsh due to a 33% reduction in the proposed concentration of sodium periodate.
The reagents used to develop the peroxidase conjugated with the monoclonal antibodies contain hydrogen peroxide, which is the substrate for the enzyme, and an appropriate chromogen, for example orthophenylene-diamine or 2,2xe2x80x2-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid, or ABTS, to give a final reaction product which is colored and soluble in the medium, or else 3,3xe2x80x2-diaminobenzidine, 3-amino-9-ethylcarbazole or 4-chloro-alpha-naphthol to give an insoluble final reaction product, or else parahydroxyphenylpropionic acid to give a fluorescent reaction product which is soluble in the medium.
In a preferred form, the kit according to the invention for the determination of antigens characteristic of the T, T4and T8 lymphocytes comprises:
a) a microtiter plate in whose wells one or more monoclonal antibodies directed against T lymphocytes have been fixed,
b1) a solution containing at least one peroxidase-labeled monoclonal antibody directed against T lymphocytes,
b2) a solution containing at least one eroxidase-labeled monoclonal antibody directed gainst CD4 antigen,
b3) a solution containing at least one peroxidase-labeled monoclonal antibody directed against CD8 antigen,
c1) a solution containing hydrogen peroxide, which is the substrate for the enzyme, in an appropriate buffer, and
c2) a solution containing the chromogen used to develop the expression of the enzyme""s activity.
Another preferred embodiment of the invention is the use of monoclonal antibodies coupled to acetyl-cholinesterase.
The acetylcholinesterase is preferably coupled to the antibody using a process derived from that described in French Patent no. 2 550 799 or a process which schematically involves the preparation of fragments of the antibody by a known technique, the modification of the enzyme by reaction with an appropriate hetero-bifunctional agent and, finally, the coupling of the resulting products. Other known processes for constructing immunoenzyme conjugates can also be used in this case.
Development of the enzyme activity specifically associated with the cell antigen recognized by the acetylcholinesterase conjugate is preferably carried out by the known technique which employs acetylthiocholine as the substrate for the enzyme and Ellman""s reagent, or 5,5xe2x80x2-dithio-2-nitrobenzoic acid, as the chromogen, in accordance with any variant suitable for the case under examination, for example the one described by Pradelles et al., Anal. Chem.,57 (1985) 1170-1173.
The chromogens cited are used as such or in the form of water-soluble salts.
The results of the determination of antigens according to the invention can be expressed in any form appropriate to the examination carried out. More particularly, these results can be expressed either as the total number of molecules of a particular antigen (for example the CD4 antigen) present in a given volume of the sample examined (for example per microliter of blood), or as the ratio of the number of molecules of one antigen to the number of molecules of another antigen in the sample examined (for example the ratio of the CD4 antigens to the CD8 antigensxe2x80x94or CD4/CD8 ratioxe2x80x94in the blood sample examined).
The number of molecules of a particular antigen in the sample examined will preferably be determined using a standard range consisting of appropriate cells or cell preparations carrying the antigen to be determined, which will have been calibrated beforehand by a known reference method. These standards will preferably consist either of cells identical in their origin to the cells which are to form the subject of the determination, or of cells of established cell lines carrying the desired antigen, or of preparations, for example membrane preparations, originating from these cells.
These standards are then treated in exactly the same way as the samples to be examined. The resulting signals are used to build up a standard range against which the signals measured with the samples to be examined are compared. The subsequent calculations are conventional.
To determine the ratio of the numbers of molecules of two antigens in the sample to be examined, it is possible to use the standard system described above and finally to calculate the desired ratio. A simpler possibility in many cases is directly to calculate the ratio of the specific signals obtained with each of the desired antigens and to correct it, if necessary, by means of known factors such as the ratio of the dimensions of the samples used; this gives the desired ratio directly.
The immunometric assay method according to the invention is simple, rapid and reproducible. Its use is totally suitable for the analysis of a large number of samples. For an understanding of its advantages compared with the other methods described, the various steps of the method according to the invention should be analyzed.
The immobilization of the cells on the solid support is the phase of the determination which usually presents the most difficulties or which is the most critical to carry out. The means often used is chemical fixation of the cells with glutaraldehyde or methanol in cups which may or may not have been treated with poly-L-lysine (F. VAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 109). However, chemical fixations performed in this way can reduce or even suppress the desired specific detection or conversely can induce the false-positive labeling of cells, which is a very serious disadvantage (DROVER and MARSHALL, J. Immunol. Methods, 1986, 90, 275-281).
Furthermore, the chemical fixation process has to be carried out in several steps: centrifugation of the cells, preparation of the fixative mixture, fixation and then washing of the fixed cells several times.
Drying of the cells at 37xc2x0 C., optionally followed by fixation with methanol in the microwells, has also been proposed (BAUMGARTEN, J. Immunol. Methods, 1986, 94, 91-98). Actually, in addition to the fact that it can degrade certain fragile antigens, drying of the cells at 37xc2x0 C. renders the pericellular plasmic membrane permeable, thereby facilitating the immunolabeling of intracyto-plasmic antigens as well as labeling of the surface antigens, which leads to troublesome backgrounds or to false-positive results when it is desired to restrict a measurement to the surface antigens.
Furthermore, the reproducibility of this process is doubtful; in fact, the settling of the cells in the assay microwells and the drying of the cells can vary between experiments. Finally, this determination is long to perform because the cell drying step alone takes more than 2 hours.
The immobilization of lymphocytic populations has also been achieved by the use of polyclonal antibodies adsorbed in microwells (STOCKER and HEUSSER, J. Immunol. Methods, 1979, 26, 87-95). Apart from permitting the immobilization of cells foreign to the single population which it is desired to analyze, polyclonal antibodies also have the disadvantage of reacting with the antigens which are to be measured by means of the labeled antibody, leading to a corresponding reduction in the signal which is finally measured.
The use of highly specific and related monoclonal antibodies adsorbed on or fixed to the solid support, and especially in the assay wells, permits exclusive capture of the desired cells, the other non-retained cell populations being removed in the course of the washing operations carried out on completion of the labeling of the antigens to be determined. Furthermore, no chemical or physical agent modifies the characteristics of the antigens in this step because the various operations for chemical or physical fixation of the cells to the support are omitted.
Thus, according to the present invention, it has been found that the immobilization of cells by monoclonal antibodies is a process which makes it possible to simplify the step for immobilizing the cells carrying the antigen to be determined, while at the same time making the results more reliable.
Immunometric assays applied to cell populations are generally carried out by labeling the cells according to an indirect method in 2 or 3 successive steps, the probe which provides the specific signal being fixed to the cells during the last step of the labeling process. In the labeling of cells in two steps, the main reagents involved use anti-immunoglobulin (anti-Ig) antibodies coupled to beta-galactosidase (COBBOLD et al., J. Immunol. Methods, 1971, 44, 125-133) or to alkaline phosphatase (HESSIAN, J. Immunol. Methods, 1986, 91, 29-34) or labeled with iodine 125 (SAVION, J. Immunol. Methods, 1987, 97, 49-56). The technique of labeling with the peroxidase-antiperoxidase reagent is carried out in 3 steps (VAN LEUVEN, 1978).
Another process, also in 3 steps, uses a monoclonal antibody specific for the antigenic marker to be analyzed, then anti-mouse Ig antibodies carrying biotin, and finally a streptavidine-peroxidase conjugate (BAUMGARTEN, 1986) or streptavidine-alkaline phosphatase conjugate (IGIETSEME et al., J. Immunol. Methods, 1987, 97, 123-131).
The process according to the invention, which comprises the simultaneous use, in a single step, of a procedure for immunocapture of whole cells without physical or chemical intervention on the cells, and of the labeling of all or some of these cells with one or more monoclonal antibodies directly carrying a radioiso-topic or enzyme probe, is the first process to permit the Quantitative determination of chosen membrane antigens on the cells themselves.
According to the invention, the direct labeling of immunologically immobilized cells permits:
simplification of the method of determination by elimination of the intermediate manipulations repeated between the successive steps of the labeling process in the case of indirect labeling: centrifugation of the cells several times, removal of the labeling reagents and resuspension;
a saving of reagents;
an improved reliability through a reduction in the number of steps and manipulations;
a time saving; and
the possibility of treating large numbers of samples at the same time, exclusively with the use of conventional equipment and apparatuses.
The incubation period for immobilization of the cell population and simultaneous direct labeling of the antigens of the cell subpopulation to be determined is short. It is less than or equal to 1 hour in the case of the determination of T lymphocytes and subpopulations of T4 and T8 lymphocytes.
After the solid support has been washed, the actual determination is carried out by using conventional apparatuses to observe a signal which is precise and simple to measure: radioactivity or light absorption or emission.
Thus, overall, the process according to the invention has numerous advantages: it is rapid, reliable, economic and simple.
The process according to the invention makes it possible to determine surface antigens of a cell population over a wide range of cell concentrations.
The sensitivity of the method in respect of the number of cells depends on the antigen density of the cell population determined. For each antigen it is possible, if desired, to define the minimum molar concentration of antigen which can be measured by the process according to the invention.
Thus, for example, when the chosen solid support is a microtiter plate and the cells examined are human lymphocytes, it has been observed that significant measurements are obtained when the number of cells analyzed is between a few hundred and about 200 000 per 200 xcexcl microwell, the lower limit being imposed by the density of the measured antigen on the cells examined and the sensitivity of the chosen detection technique, while the upper limit depends essentially on the size and geometry of the solid support. The same applies in the case where the solid support consists of tubes.
It has been verified that the signals recorded (radioactivity count or photometric measurements) make it possible to obtain satisfactory uniform calibration curves as a function of the number of cells used, under the customary handling conditions.
Furthermore, the sensitivity of the method can be improved, if necessary, by the simultaneous fixation, to one and the same surface antigen, of several different monoclonal antibodies specific for several different epitopes of the same antigen. This has been verified by determination of the CD4 antigens on cells of the Ichikawa line (human T line), where the OKT4 and ST4 antibodies labeled with iodine 125 were used simultaneously. The measured signal increased by about 50% relative to the signals obtained with each of the anti-CD4 antibodies used by itself.